Corynebacterium glutamicum as a platform strain for the production of a broad variety of terpenoids

Corynebacterium glutamicum is a natural carotenoid producing bacterium used in the million-ton-scale amino acid biotechnology that has been engineered for isoprenoid production1. The native membrane-bound carotenoid decaprenoxanthin is a rare C50 carotenoid. Volatile terpenoids such as valencene2 and patchoulol3 could be produced upon deletion of the first step of the specific carotenoid pathway and heterologous expression of the FPP synthase gene ispA from E. coli and terpene synthases from plant origin. However, these strains produced a yet unidentified carotenoid and only when all carotenoid biosynthetic genes were deleted, a colorless strain resulted. Expressing a codon optimized ADS from Artemisia annua in the white strain, amorphadiene, the volatile precursor for artemisinin was produced. For production of volatile terpenoids a dodecane overlay was used, a condition in which C. glutamicum benefits from its robust myco-membrane. Recently, we showed production of membrane-bound carotenoids with different length and/or cyclization status: bicyclic C50 sarcinaxanthin4, bicyclic C40 astaxanthin5, the linear lycopene6 and the linear C50 bisanhydrobacterioruberin7. This indicated that the C. glutamicum myco-membrane accepts these linear and bicyclic carotenoids. Please click Additional Files below to see the full abstract

Metabolic engineering of Corynebacterium glutamicum for the sustainable production of aromatic compounds

Burgardt A. Metabolic engineering of Corynebacterium glutamicum for the sustainable production of aromatic compounds. Bielefeld: Universität Bielefeld; 2022

Utilization of a wheat sidestream for 5-aminovalerate production by Corynebacterium glutamicum

Burgardt A, Prell C, Wendisch VF. Utilization of a wheat sidestream for 5-aminovalerate production by Corynebacterium glutamicum. Frontiers in Bioengineering and Biotechnology. 2021;9: 732271

Fermentative production of ʟ-2-hydroxyglutarate by engineered Corynebacterium glutamicum via pathway extension of ʟ-lysine biosynthesis

Prell C, Burgardt A, Meyer F, Wendisch VF. Fermentative production of ʟ-2-hydroxyglutarate by engineered Corynebacterium glutamicum via pathway extension of ʟ-lysine biosynthesis. Frontiers in Bioengineering and Biotechnology. 2021;8: 630476.L-2-hydroxyglutarate (L-2HG) is a trifunctional building block and highly attractive for the chemical and pharmaceutical industries. The natural L-lysine biosynthesis pathway of the amino acid producer Corynebacterium glutamicum was extended for the fermentative production of L-2HG. Since L-2HG is not native to the metabolism of C. glutamicum metabolic engineering of a genome-streamlined L-lysine overproducing strain was required to enable the conversion of L-lysine to L-2HG in a six-step synthetic pathway. To this end, L-lysine decarboxylase was cascaded with two transamination reactions, two NAD(P)-dependent oxidation reactions and the terminal 2-oxoglutarate-dependent glutarate hydroxylase. Of three sources for glutarate hydroxylase the metalloenzyme CsiD from Pseudomonas putida supported L-2HG production to the highest titers. Genetic experiments suggested a role of succinate exporter SucE for export of L-2HG and improving expression of its gene by chromosomal exchange of its native promoter improved L-2HG production. The availability of Fe2+ as cofactor of CsiD was identified as a major bottleneck in the conversion of glutarate to L-2HG. As consequence of strain engineering and media adaptation product titers of 34 ± 0 mM were obtained in a microcultivation system. The glucose-based process was stable in 2 L bioreactor cultivations and a L-2HG titer of 3.5 g L−1 was obtained at the higher of two tested aeration levels. Production of L-2HG from a sidestream of the starch industry as renewable substrate was demonstrated. To the best of our knowledge, this study is the first description of fermentative production of L-2HG, a monomeric precursor used in electrochromic polyamides, to cross-link polyamides or to increase their biodegradability

Dynamic Co-Cultivation Process of Corynebacterium glutamicum Strains for the Fermentative Production of Riboflavin.

Residual streams from lignocellulosic processes contain sugar mixtures of glucose, xylose, and mannose. Here, the industrial workhorse Corynebacterium glutamicum was explored as a research platform for the rational utilization of a multiple sugar substrate. The endogenous manA gene was overexpressed to enhance mannose utilization. The overexpression of the xylA gene from Xanthomonas campestris in combination with the endogenous xylB gene enabled xylose consumption by C. glutamicum. Furthermore, riboflavin production was triggered by overexpressing the sigH gene from C. glutamicum. The resulting strains were studied during batch fermentations in flasks and 2 L lab-scale bioreactors separately using glucose, mannose, xylose, and a mixture of these three sugars as a carbon source. The production of riboflavin and consumption of sugars were improved during fedbatch fermentation thanks to a dynamic inoculation strategy of manA overexpressing strain and xylAB overexpressing strain. The final riboflavin titer, yield, and volumetric productivity from the sugar mixture were 27 mg L−1 , 0.52 mg g−1 , and 0.25 mg L−1 h −1 , respectively. It reached a 56% higher volumetric productivity with 45% less by-product formation compared with an equivalent process inoculated with a single strain overexpressing the genes xylAB and manA combined. The results indicate the advantages of dynamic multi strains processes for the conversion of sugar mixtures

Rational Engineering of Non-Ubiquinone Containing Corynebacterium glutamicum for Enhanced Coenzyme Q10 Production

Burgardt A, Pelosi L, Hajj Chehade M, Wendisch VF, Pierrel F. Rational Engineering of Non-Ubiquinone Containing Corynebacterium glutamicum for Enhanced Coenzyme Q10 Production. Metabolites. 2022;12: 428

Recent advances in the metabolic pathways and microbial production of coenzyme Q

Pierrel F, Burgardt A, Lee J-H, Pelosi L, Wendisch VF. Recent advances in the metabolic pathways and microbial production of coenzyme Q . World Journal of Microbiology and Biotechnology. 2022;38(4): 58.Coenzyme Q (CoQ) serves as an electron carrier in aerobic respiration and has become an interesting target for biotechnological production due to its antioxidative effect and benefits in supplementation to patients with various diseases. Here, we review discovery of the pathway with a particular focus on its superstructuration and regulation, and we summarize the metabolic engineering strategies for overproduction of CoQ by microorganisms. Studies in model microorganisms elucidated the details of CoQ biosynthesis and revealed the existence of multiprotein complexes composed of several enzymes that catalyze consecutive reactions in the CoQ pathways of Saccharomyces cerevisiae and Escherichia coli. Recent findings indicate that the identity and the total number of proteins involved in CoQ biosynthesis vary between species, which raises interesting questions about the evolution of the pathway and could provide opportunities for easier engineering of CoQ production. For the biotechnological production, so far only microorganisms have been used that naturally synthesize CoQ10 or a related CoQ species. CoQ biosynthesis requires the aromatic precursor 4-hydroxybenzoic acid and the prenyl side chain that defines the CoQ species. Up to now, metabolic engineering strategies concentrated on the overproduction of the prenyl side chain as well as fine-tuning the expression of ubi genes from the ubiquinone modification pathway, resulting in high CoQ yields. With expanding knowledge about CoQ biosynthesis and exploration of new strategies for strain engineering, microbial CoQ production is expected to improve

Dynamic co-cultivation process of Corynebacterium glutamicum strains for the fermentative production of riboflavin

Perez F, Burgardt A, Kallmann DR, Wendisch VF, Bar N. Dynamic co-cultivation process of Corynebacterium glutamicum strains for the fermentative production of riboflavin. Fermentation. 2021;7(1): 11

Fermentative production of halogenated tryptophan derivatives with Corynebacterium glutamicum overexpressing tryptophanase or decarboxylase genes

Kerbs A, Burgardt A, Veldmann K, Schäffer T, Lee J-H, Wendisch VF. Fermentative production of halogenated tryptophan derivatives with Corynebacterium glutamicum overexpressing tryptophanase or decarboxylase genes. ChemBioChem. 2022;23: e202200007.The aromatic amino acid tryptophan serves as a precursor for many valuable compounds such as neuromodulators, indoleamines and indole alkaloids. In this work, tryptophan biosynthesis was extended by halogenation followed by decarboxylation to the respective tryptamines or cleavage to the respective indoles. Either the tryptophanase genes tnaAs from E. coli and Proteus vulgaris or the aromatic amino acid decarboxylase genes AADCs from Bacillus atrophaeus, Clostridium sporogenes, and Ruminococcus gnavus were expressed in C. glutamicum strains producing (halogenated) tryptophan. Regarding indoles, final titers of 16 mg L-1 7-Cl-indole and 23 mg L-1 7-Br-indole were attained. Tryptamine production led to a much higher titer of 2.26 g L-1 upon expression of AADC from B. atrophaeus. AADCs were shown to be active with halogenated tryptophan in vitro and in vivo and supported production of 0.36 g L-1 7-Br-tryptamine with a volumetric productivity of 8.3 mg L-1 h-1 in a fed-batch fermentation

Can DapC be the missing aminotransferease in the arogenate route of L-tyrosine biosynthesis in Corynebacterium glutamicum?

Kaya FEA, Kurpejovic E, Burgardt A, Wendisch VF, Akbulut BS. Can DapC be the missing aminotransferease in the arogenate route of L-tyrosine biosynthesis in Corynebacterium glutamicum? In: Supplement: The Biochemistry Global Summit, 25th IUBMB Congress, 46th FEBS Congress, 15th PABMB Congress, July 9–14, 2022, Lisbon, Portugal. FEBS Open Bio . Vol 12. Hoboken: Wiley; 2022: 180-181